The primary goal of this proposal is to study the structure and function of 4- oxalocrotonate tautomerase (4-OT) by exploiting the advantages afforded by total chemical synthesis. The research outlined here is centered on the synthesis and characterization of 4-OT analogs designed to address specific questions about the catalytic mechanism of this highly efficient enzyme. The structural and mechanistic implications of a hydrophobic pocket surrounding the active site will be investigated along with those factors important for substrate binding. These studies will involve systematically varying the side chain functionalities of 3 catalytically important residues: Arg1, Arg39, and Pro1. The 4-OT analogs generated here will be subject to kinetic analysis and characterized by circular dichroism and native electrospray ionization mass spectrometry. The results of these experiments should lead to a better understanding of the factors that influence enzyme catalysis. Furthermore, a synthetic strategy for covalently linking specific subunits in 4-OT's active hexameric complex is also proposed. The ability to assemble active enzyme complexes using such a modular approach will permit the total chemical synthesis of larger proteins and allow the construction of multimeric proteins with specific mutations in individual subunits.